Role of cyclooxygenase isoforms in prostacyclin biosynthesis and murine prehepatic portal hypertension.

Portal hypertension (PHT) is a common complication of liver cirrhosis and significantly increases morbidity and mortality. Abrogation of PHT using NSAIDs has demonstrated that prostacyclin (PGI(2)), a direct downstream metabolic product of cyclooxygenase (COX) activity, is an important mediator in the development of experimental and clinical PHT. However, the role of COX isoforms in PGI(2) biosynthesis and PHT is not fully understood. Prehepatic PHT was induced by portal vein ligation (PVL) in wild-type, COX-1(-/-), and COX-2(-/-) mice treated with and without COX-2 (NS398) or COX-1 (SC560) inhibitors. Hemodynamic measurements and PGI(2) biosynthesis were determined 1-7 days after PVL or sham surgery. Gene deletion or pharmacological inhibition of COX-1 or COX-2 attenuated but did not ameliorate PGI(2) biosynthesis after PVL or prevent PHT. In contrast, treatment of COX-1(-/-) mice with NS398 or COX-2(-/-) mice with SC560 restricted PGI(2) biosynthesis and abrogated the development of PHT following PVL. In conclusion, either COX-1 or COX-2 can mediate elevated PGI(2) biosynthesis and the development of experimental prehepatic PHT. Consequently, PGI(2) rather then COX-selective drugs are indicated in the treatment of PHT. Identification of additional target sites downstream of COX may benefit the >27,000 patients whom die annually from cirrhosis in the United States alone.

Inhibition of alcohol dehydrogenase blocks enhanced Gi-protein expression following ethanol treatment in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. DESIGN: Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. METHODS: These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. RESULTS: Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. CONCLUSIONS: These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.

Functional venous anatomy for right-lobe grafting and techniques to optimize outflow.

Right-lobe living donor liver transplantation has emerged as an alternative to cadaveric transplantation. An appreciation of the unique anatomy and behavior of the right lobe has emerged and has precipitated technical modifications. Living donors underwent right lobectomy, including preservation of significant inferior hepatic veins. The parenchyma was divided following a plane approximating the right border of the posterior two thirds of the midhepatic vein (MHV), but deviating anteriorly to include the distal one third of the MHV with the graft. Large venous tributaries from segment VIII were preserved. Anastomosis in the recipient was accomplished by means of complete cavoplasty. Significant inferior veins, tributaries to the MHV, and the distal portion of the MHV were reconstructed when technically possible. Forty-eight right-lobe resections and transplantations were performed in the manner described. There were no donor complications attributable to the technique. Forty-six of the 48 recipients are alive, and 44 of the 46 surviving patients have their original graft. Venous tributaries from segment VIII and/or the distal portion of the MHV were reconstructed in only 3 patients. Outflow obstruction was recognized intraoperatively in 2 patients; 1 patient had a caval web excised and the other patient required revision of the main anastomosis. Neither organ was lost. There were no other significant venous complications. The incidence of ascites was the same as that in recipients of whole organs. These methods of parenchymal transection and venous reconstruction resulted in a low rate of complications. The wide anastomosis and collateral pathways between the MHV and right hepatic vein seem to be more critical than reconstruction of tributaries from segment VIII or the distal MHV.

Endothelial dysfunction in cirrhosis and portal hypertension.

Portal hypertension (PHT) is a common clinical syndrome associated with chronic liver diseases; it is characterized by a pathological increase in portal pressure. Pharmacotherapy for PHT is aimed at reducing both intrahepatic vascular tone and elevated splanchnic blood flow. Due to the altered hemodynamic profile in PHT, dramatic changes in mechanical forces, both pressure and flow, may play a pivotal role in controlling endothelial and vascular smooth muscle cell signaling, structure, and function in cirrhotics. Nitric oxide, prostacyclin, endothelial-derived contracting factors, and endothelial-derived hyperpolarizing factor are powerful vasoactive substances released from the endothelium in response to both humoral and mechanical stimuli that can profoundly affect both the function and structure of the underlying vascular smooth muscle. This review will examine the contributory role of hormonal- and mechanical force-induced changes in endothelial function and signaling and the consequence of these changes on the structural and functional response of the underlying vascular smooth muscle. It will focus on the pivotal role of hormonal and mechanical force-induced endothelial release of vasoactive substances in dictating the reactivity of the underlying vascular smooth muscle, i.e., whether hyporeactive or hyperreactive, and will examine the extent to which these substances may exert a protective and/or detrimental influence on the structure of the underlying vascular smooth muscle in both a normal hemodynamic environment and following hemodynamic perturbations typical of PHT and cirrhosis. Finally, it will discuss the intracellular processes that regulate the release/expression of these vasoactive substances and that control the transformation of this normally protective cell to one that may promote the development of vasculopathy in PHT.

Cytokine-responsive gene-2/IFN-inducible protein-10 expression in multiple models of liver and bile duct injury suggests a role in tissue regeneration.

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl(4) treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl(4), D-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl(4) injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.

Endothelial cells inhibit flow-induced smooth muscle cell migration: role of plasminogen activator inhibitor-1.

BACKGROUND: The endothelium may play a pivotal role in hemodynamic force-induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. METHODS AND RESULTS: Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm(2)) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264+/-26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1-/- ECs, flow increased wild-type SMC migration (226+/-25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration. CONCLUSIONS: Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.

Management of massive retroperitoneal hemorrhage from an adrenal tumor.

Spontaneous massive retroperitoneal hemorrhage from an adrenal gland is a rare event. A thoughtful and meticulous approach to such a patient, with appropriate diagnostic studies, ICU and surgical care are essential for patient survival. In patients with active bleeding, angiographic embolization is a valuable adjunct to achieve hemostasis, to allow for further work-up of the adrenal tumor, and an improved subsequent oncologic resection. Hemodynamically unstable patients, however, may require supportive transfusions in the intensive care unit, potential embolization if deemed feasible, or urgent surgical exploration. If possible, however, the acute surgical removal of an adrenal tumor within a large retroperitoneal hematoma should be avoided, as under such conditions a proper oncologic resection may not be possible. The possibility of a pheochromocytoma must always be entertained. Early recognition and treatment of patients with presumed adrenal insufficiency may decrease patient morbidity and mortality.

Laparoscopic donor nephrectomy with a 23-hour stay: a new standard for transplantation surgery.

OBJECTIVE: To determine the outcomes associated with laparoscopic donor nephrectomy (LDN) performed as a 23-hour day surgery protocol. SUMMARY BACKGROUND DATA: Living donor renal transplantation is associated with immediate graft function and prolonged graft survival. The recent application of laparoscopic technology to living renal donation has refocused attention on the advantages of this donor source. LDN is associated with decreased donor pain, length of stay, time out of work, and opportunity costs. The authors hypothesized that LDN would be a viable 23-hour stay procedure. METHODS: All living donation procedures since May 1998 have been performed with LDN. The 23-hour protocol was initiated in January 1999. LDNs performed from May 1998 to December 1998 and traditional open donor nephrectomy procedures from May 1996 to May 1998 served as historical controls. The following variables were examined: donor sex, related versus nonrelated donation, operative time, blood loss, length of stay, time out of work, recipient and donor serum creatinine levels, hospital charges, and complications. RESULTS: The 23-hour LDN protocol was associated with high degrees of donor satisfaction. Thirty-six of the 41 donors were discharged within 23 hours; 1 was readmitted. Time out of work was equivalent to that of the control group; graft function was identical among all three comparison groups. Hospital charges were equivalent between the control group and the open nephrectomy group and were significantly decreased in the 23-hour group. CONCLUSIONS: Laparoscopic donor nephrectomy can be performed as a 23-hour stay procedure without incurring additional complications or donor dissatisfaction. By further decreasing opportunity costs and disincentives for donation, LDN may increase the number of potential donors appearing for evaluation.

Pulmonary expression of iNOS and HO-1 protein is upregulated in a rat model of prehepatic portal hypertension.

Portal hypertension is associated with a wide range of pulmonary pathophysiologies, ranging from portopulmonary hypertension to hepatopulmonary syndrome. Although the clinical and pathological features of pulmonary dysfunction in this setting have been extensively characterized, the underlying biology is not well understood. Specifically, the role of mediators that regulate mesenteric vascular hemodynamics in portal hypertension, such as nitric oxide and endothelin, have not been studied in the lung. Using a rat model of prehepatic portal hypertension with preserved hepatic function, we examined pulmonary elaboration of endothelial nitric oxide synthase (NOS), inducible NOS, heme oxygenase- 1 (HO-1), heme oxygenase-2 (HO-2), endothelin-1 mRNA, and protein. In comparison to sham controls, portal hypertensive animals exhibited significantly increased pulmonary iNOS and HO-1 mRNA and protein. Cyclic GMP was significantly increased in portal hypertensive lung tissue, suggesting activation of guanylyl cyclase by the endproducts of iNOS and/or HO-1 activity. Using immunohistochemical analysis, iNOS expression was localized to the vascular endothelium, while HO-1 localized to bronchiolar epithelium and macrophages. These results suggest that production of nitric oxide and carbon monoxide may contribute to the pulmonary pathology associated with portal hypertension.

Potential mechanisms for cardiovascular protective effect of ethanol.

Epidemiological studies demonstrate a significant protective effect of moderate alcohol consumption on the incidence of cardiovascular diseases which accounts for the majority of deaths in the Western world. In this review, possible mechanisms to explain the cardioprotective effect of ethanol are discussed. While the prevailing theory supported by a number of clinical and animal studies indicates that the ability of ethanol to elevate serum high-density lipoprotein (HDL) cholesterol levels is an important mechanism in ameliorating cardiovascular disease, other mechanisms whereby ethanol could exert its beneficial effect have been proposed. Namely, its ability to affect platelet function and endothelial cell and vascular smooth muscle cell function (In this review, the terms alcohol and ethanol are used interchangeably).

Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. METHODS: These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. RESULTS: Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. CONCLUSIONS: These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.

Phenotype dictates the growth response of vascular smooth muscle cells to pulse pressure in vitro.

The objective of this study was to determine the effect of phenotype on pulse pressure-induced signaling and growth of vascular smooth muscle cells in vitro. Using a perfused transcapillary culture system, cells were exposed to increases in pulsatile flow and hence pulse pressure and maintained for 72 h before cells were harvested. Cell proliferation was determined by cell number, DNA synthesis, and proliferating cell nuclear antigen expression. Mitogen-activated protein kinase (MAPK) levels were determined by immunoblot and kinase activity by phosphorylation of myelin basic protein. Cell phenotype was determined by immunoblot and immunocytofluorescence using antisera specific for the differentiation markers alpha-actin, myosin, calponin, osteopontin, and phospholamban. In cells that highly expressed these differentiation markers, there was a significant increase in cell growth in response to chronic increases in pulse pressure without a significant change in MAPK activity in these cells. In contrast, in cells that weakly expressed SMC differentiation markers, there was a significant decrease in cell growth concomitant with a significant decrease in MAPK signaling in these cells. We conclude that SMC phenotype dictates the growth response of SMC to mechanical force in vitro.

Ethanol inhibits basal and flow-induced vascular smooth muscle cell migration in vitro.

BACKGROUND: Alcohol consumption protects against coronary heart disease by as yet unclear mechanisms. The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell (SMC) migration which plays an important role in the pathogenesis of atherosclerosis. MATERIALS AND METHODS: Cultures of human SMC under static (no flow) or pulsatile flow conditions (perfused transcapillary culture system) were pretreated in the absence or presence of ethanol (EtOH) whereupon their random migration (chemokinesis) was assessed by Transwell assay. RESULTS: Ethanol pretreatment (24 h) dose dependently inhibited migration of HuSMC from static cultures with a maximal inhibition of 60.8 +/- 4.4% observed at 40-80 mM, in the absence of any effect on cell adhesion or cell viability as assessed by trypan blue exclusion. In HuSMC exposed to pulsatile flow (0.3 to 25 ml/min, 24 h), there was a flow-dependent increase in migration ranging from a 1.3 +/- 0.16- to 2.67 +/- 0.26-fold increase, compared to static cells, concomitant with a significant increase in urokinase-type plasminogen activator (uPA) mRNA levels. Ethanol pretreatment (20-80 mM, 24 h) dose dependently inhibited the flow-induced increase in SMC migration but did not affect uPA mRNA expression. CONCLUSIONS: The inhibitory effect of ethanol on basal and flow-stimulated SMC migration may be relevant to its cardiovascular effects in vivo.

Ethanol enhances basal and flow-stimulated nitric oxide synthase activity in vitro by activating an inhibitory guanine nucleotide binding protein.

The aim of this study was to determine the effect of ethanol on endothelial nitric oxide synthase (eNOS), the enzyme responsible for the production of the important vasoactive agent nitric oxide. The effect of ethanol (0.8-160 mM) on both basal and flow-stimulated eNOS activity was determined using cultured bovine aortic endothelial cells (EC). In "static" EC ethanol dose-dependently increased basal eNOS activity with a maximum response (approximately 2.0-fold increase) achieved at 40 mM in the absence of any effect on cell viability or nitric oxide synthase protein expression. Pertussis toxin (PTX) pretreatment significantly inhibited the ethanol-induced increase in basal eNOS activity. EC exposed to steady laminar flow exhibited a flow- and time-dependent increase in eNOS activity. Ethanol significantly enhanced the laminar flow-induced eNOS response from 0.62 +/- 0.1 to 1.06 +/- 0. 06 pmol [14C]citrulline/mg/min, a response that was inhibited by PTX. PTX-catalyzed ribosylation of Gialpha substrates, an index of G-protein functional activity, was increased in laminar flow-exposed EC compared with static controls and was further enhanced by ethanol treatment. Likewise, EC exposed to low ( approximately 0.5 dynes/cm2) and high ( approximately 12 dynes/cm2) pulsatile flow demonstrated increased eNOS activity, an effect that was associated with increased PTX-catalyzed ribosylation of Gialpha substrates. Ethanol enhanced the low flow response in a PTX-sensitive manner. These data demonstrate a stimulatory effect of ethanol on basal and flow-stimulated eNOS activity, mediated in part by a mechanism involving a PTX-sensitive G protein.

Sustained pulsatile flow regulates endothelial nitric oxide synthase and cyclooxygenase expression in co-cultured vascular endothelial and smooth muscle cells.

This study addresses the effect of sustained increased pulsatile flow on nitric oxide synthase (NOS) and cyclooxygenase (Cox) expression and activity in co-cultured endothelial cells (EC) and vascular smooth muscle cells (SMC). Using a perfused transcapillary co-culture system which permits the chronic exposure of cultured EC and SMC to physiological shear stresses, co-cultures were exposed to step-wise increases in flow up to: (i) 2 ml/min (low flow: 0.5 dyn/cm2): or (ii) 44 ml/min (high flow: 15 dyn/cm2) and maintained for 72 h before SMC and EC were harvested separately. There was no NOS activity or protein expression in co-cultured SMC under flow conditions. There was a significant increase in eNOS activity in co-cultured EC under high flow conditions, compared to low flow, which correlated with an increase in eNOS expression and mRNA levels. The flow-induced increase in eNOS activity was potentiated by indomethacin treatment, suggesting a modulatory role for a cyclooxygenase product. Prostacyclin levels in co-culture perfusate were significantly elevated under high flow conditions. While both co-cultured EC and SMC expressed cyclooxygenase (Cox-I and Cox-II), they were differentially regulated by pulsatile flow, EC Cox-I and Cox-II protein expression were both decreased. Indomethacin treatment increased the expression of both Cox-I and Cox-II in co-cultured SMC under high flow conditions. We conclude that sustained increases in pulsatile flow maintain elevated eNOS and Cox protein expression and activity in EC while decreasing Cox expression in co-cultured SMC. These data suggest that regulation of these pathways may contribute to flow-induced vascular remodeling in vivo.

Effect of pulse pressure on vascular smooth muscle cell migration: the role of urokinase and matrix metalloproteinase.

Plasminogen activator (PA) expression plays an important role in smooth muscle cell (SMC) migration and may therefore contribute to mechanical force-induced arterialization of vein grafts. The aim of this study was to determine whether pulse pressure due to pulsatile flow modulates SMC migration via urokinase (u-PA)-dependent mechanisms. Using a perfused transcapillary culture system, human umbilical vein SMC were exposed to pulse pressures (0-56 mmHg), in the absence or presence of human umbilical vein endothelial cells (EC) by varying pulsatile flow rates (0 ml/min to 25 ml/min). SMC cultured in the absence of EC increased their migration following exposure to increased pulse pressure (248+/-14%). Both u-PA and matrix metallo-proteinase 1 (MMP-1) expression was significantly elevated in SMC exposed to pressure as compared to static controls. The role of proteases in the pulse pressure-induced enhancement of SMC migration was confirmed following pretreatment with aprotinin, an anti u-PA antibody and metalloproteinase inhibitors (181+/-14% for aprotinin vs. 256+/-25% for control, 108+/-4% for anti-u-PA antibody vs. 233+/-17% for non-immune IgG, and 114+/-9% for BB-94, 105+/-7% for BB-3103 vs. 222+/-5% for control). Using SMC derived from u-PA gene knock-out mice, the SMC migratory response to increased pulse pressure was completely inhibited despite a significant increase in MMP expression in these cells. These results suggest that pulse pressure due to pulsatile flow induces SMC migration in vitro via u-PA and MMP-dependent mechanisms. Moreover, u-PA gene deletion results in blunting of pressure-induced SMC migration despite the endogenous upregulation of metalloproteinase. Modulation of u-PA expression by pressure may thus represent an important mechanism whereby hemodynamic forces regulate smooth muscle cell migration.

Nonanticoagulant heparin prevents coronary endothelial dysfunction after brief ischemia-reperfusion injury in the dog.

BACKGROUND: Coronary endothelial dysfunction after brief ischemia-reperfusion (IR) remains a clinical problem. We investigated the role of heparin and N-acetylheparin, a nonanticoagulant heparin derivative, in modulating coronary endothelial function after IR injury, with an emphasis on defining the role of the nitric oxide (NO)-cGMP pathway in the heparin-mediated effect. METHODS AND RESULTS: Male mongrel dogs were surgically instrumented, and the effects of both bovine heparin and N-acetylheparin on coronary endothelial vasomotor function, expressed as percent change from baseline flow after acetylcholine challenge, were studied after 15 minutes of regional ischemia of the left anterior descending artery (LAD) followed by 120 minutes of reperfusion. In dogs treated with placebo (saline), coronary vasomotor function was significantly (P

Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis.

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.

Enhanced Gi-protein-mediated mitogenesis following chronic ethanol exposure in a rat model of experimental hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is associated with increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the effects of chronic ethanol exposure on the expression and function of adenylyl cyclase (AC)-linked G-proteins (Gs and Gi) and growth in experimental HCC. G-protein expression and function was determined by immunoblot in the hepatic tumorigenic H4IIE cell line and isolated cultured hepatocytes in the absence or presence of ethanol (5-100 mmol/L). Chronic exposure (24 hours) to ethanol dose-dependently increased Gialpha1/2 expression in the H4IIE cell line, but not in cultured hepatocytes. Gsalpha-protein expression remained unchanged in both H4IIE cells and cultured hepatocytes following ethanol treatment. In addition, ethanol directly activated a Gi-protein, because pertussis toxin (PTx)-catalyzed, adenosine diphosphate (ADP)-dependent ribosylation of Gialpha substrates decreased following ethanol treatment. The increased functional activity of Gialpha1/2-protein expression was confirmed by demonstrating that ethanol dose-dependently inhibited basal and stimulated AC activity in H4IIE cells, while not significantly altering basal AC activity in isolated cultured hepatocytes. Furthermore, while ethanol had no significant effect on basal mitogenesis in H4IIE cells or hepatocytes, increased mitogenesis caused by direct Gialpha-protein stimulation (mastoparan M7; 10-5,000 nmol/L) was further enhanced in the presence of ethanol, an effect that was completely blocked following Gi-protein inhibition (PTx; 100 ng/mL). In contrast, activation of Gi-proteins using M7 failed to alter cellular mitogenesis in isolated cultured hepatocytes, whether in the absence or presence of ethanol. Finally, analysis of mitogen-activated protein kinase (MAPK) activity demonstrated that chronic ethanol treatment further enhanced Gi-protein-stimulated MAPK activity in hepatic tumorigenic cells. In conclusion, these data demonstrate that ethanol enhances cellular mitogenesis in experimental HCC as a result of, at least in part, a Gi-MAPK-dependent pathway. Furthermore, this effect may be caused by ethanol's direct up-regulation of the expression and activity of Gi-proteins in HCC.